Synthesis and biophysical studies of oligonucleotides containing hydroxamate linkages

Article abstract of DOI:10.1081/NCN-100002513

Novel thymidine dimers containing hydroxamate linkages were synthesized, incorporated into oligonucleotide sequences and studied their hybridization properties against complementary DNA and RNA targets.

Full text of DOI:10.1081/NCN-100002513

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SYNTHESIS AND BIOPHYSICAL STUDIES OF
OLIGONUCLEOTIDES CONTAINING HYDROXAMATE
LINKAGES
Kanda S. Ramasamy a , Vesna Stoisavljevic a & Johnson Y. N. Lau a
a Medicinal Chemistry Division , ICN Pharmaceuticals, Inc. , 3300 Hyland Avenue, Costa
Mesa, California, 92626, U.S.A.
Published online: 07 Feb 2007.
To cite this article: Kanda S. Ramasamy , Vesna Stoisavljevic & Johnson Y. N. Lau (2001) SYNTHESIS AND BIOPHYSICAL
STUDIES OF OLIGONUCLEOTIDES CONTAINING HYDROXAMATE LINKAGES, Nucleosides, Nucleotides and Nucleic Acids,
20:4-7, 1173-1176, DOI: 10.1081/NCN-100002513
To link to this article: http://dx.doi.org/10.1081/NCN-100002513
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 1173–1176 (2001)
SYNTHESIS AND BIOPHYSICAL STUDIES
OF OLIGONUCLEOTIDES CONTAINING
HYDROXAMATE LINKAGES
Kanda S. Ramasamy,∗ Vesna Stoisavljevic, and Johnson Y. N. Lau
Medicinal Chemistry Division, ICN Pharmaceuticals, Inc.,
3300 Hyland Avenue, Costa Mesa, California 92626
ABSTRACT
Novel thymidine dimers containing hydroxamate linkages were synthesized,
incorporated into oligonucleotide sequences and studied their hybridization
properties against complementary DNA and RNA targets.
Inhibition of gene expression with antisense oligonucleotides by binding to
mRNA in a sequence specific manner has become an attractive method for the
treatment of viral diseases, cancer, and for the study of genetic disorders (1–3). As
drug candidates, they should inhibit the translation process via duplex formation
and/or able to activate RNase H to cleave the target message after hybridization.
In addition, antisense oligonucleotides should possess in vivo nuclease stability,
adequate binding affinity to the target mRNA, and the ability to reach target cells
and tissues. So far, chemical modifications of oligonucleotides have resulted in in-
creased solubility, nuclease stability, cellular uptake, binding properties and RNase
H activation (4). However, the quest for new and novel modified oligonucleotides
with improved properties is growing.
In this communication we report the synthesis of a novel class of thymi-
dine dimers 15 and 16, incorporation into oligonucleotide sequences and their hy-
bridization studies against natural DNA/RNA. The hydroxamate dimers 15 and
16 were synthesized as shown in Scheme 1. The known aldehyde 4 (5) was
coupled with O-methylhydroxylamine hydrochloride in the presence of pyridine
∗Corresponding author.
1173
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C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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Scheme 1.
at 100◦C for 12 h to give oxime 5 in 99% yield. Reduction of 5 with sodium
cyanoborohydride in dry methanol containing acetic acid for 6 h at room tempera-
ture provided 6. Aldehyde 7 (6) was oxidized to the corresponding carboxylic acid
8 with sodium chlorite using 2-methyl-2-butene as HOCl scavenger (7). Esterifi-
cation of 8 with benzyl alcohol using water soluble carbodiimide (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride, EDC) (8) afforded benzyl ester
9 in 87% yield. Desilylation of 9 followed by dimethoxytritylation (9) and base
hydrolysis provided second building block 12. Carboxylic acid 12 was activated
with O-(1H-benzotriaol-1-yl)-N,N,Nꢁ,Nꢁ-tetramethyluronium hexafluoroborate
(HBTU) and N-hydroxybenzotriazole (10) and then coupled with hydroxylamine
6 at room temperature to give a dimer 13 in 63% yield. Removal of the silyl
protective group from 13 with tetra-n-butylammonium fluoride and subsequent
phosphitylation (11) gave the target phosphoramidite dimer 15 (12). The second

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Table 1. Tm values of Oligonucleotides Containing O-Methylhydroxamate Linkagea
Tm (◦C)
ꢀTm/mod (◦C)
Sequence
DNA
RNA
DNA
RNA
5ꢁ GAA GCC ATC AAG CAG GAA 3ꢁ
5ꢁ TTC CTG CTT GAT GGC TTC 3ꢁ
5ꢁ TTC CTG CTT GAT GGC ttC 3ꢁ
5ꢁ ttC CTG CTT GAT GGC TTC 3ꢁ
5ꢁ ttC CTG CTT GAT GGC ttC 3ꢁ
5ꢁ TTC CTG Ctt GAT GGC TTC 3ꢁ
5ꢁ ttC CTG Ctt GAT GGC ttC 3ꢁ
5ꢁ TTC CTG Ctt GAT GGC ttC 3ꢁ
61.07
61.42
60.75
61.33
60.25
60.31
60.26
63.88
64.13
64.03
64.18
63.03
63.07
62.97
0.2
−0.2
0.1
−0.4
−0.1
−0.2
0.2
0.1
0.1
−0.4
−0.1
−0.2
aTm is the temperature at the midpoint of the melting curve; The concentrations are as
follows: Oligomer strands, 2 µM each. Melting temperatures (Tm) were determined (13)
by measuring change in absorbance at 260 nm (cuvette, 1-mm path length) as a function
of temperature in sodium phosphate buffer (10 mM, pH 7.0) containing 100 mM NaCl and
0.1 mM EDTA. All the values are averaged from at least three experiments. The letters “tt”
denote thymidine dimer containing hydroxamate linkages.
dimer 16 was also prepared by using the same methodology depicted in scheme 1
(60% overall yield) and substituting O-benzylhydroxylamine hydrochloride for
O-methylhydroxylamine hydrochloride during the formation of 5.
Incorporation of the dimers 15 and 16 (12) into oligonucleotide sequences
was accomplished using ABI 394 DNA synthesizer and protocol (14), and the cou-
pling efficiency was found to be higher than 95%. The binding behavior of the mod-
ified oligonucleotides was assayed by examining their ultraviolet (UV) absorbance
verses temperature profiles. An 18-mer oligonucleotide (5ꢁ-TTCCTGCTTGATGG-
CTTC-3ꢁ) was modified with 15 and 16 at different locations and hybridized to
complementary DNA or RNA. Melting temperatures of the duplexes formed be-
tween oligonucleotides containing the hydroxamate linkages and their DNA and
RNA complementary strands are summarized in Table 1. This study suggests that
a smaller substitution (CH3 group) on the hydroxamate dimer 15 did not affect
duplex stability. On the other hand, dimer 16 with a bulky substitution (benzyl)
in oligonucleotide sequence decreases (data not shown) its ability to form stable
duplexes with complementary DNA/RNA. Interestingly, compared to unmodified
oligonucleotides, incorporation of hydroxamate modified dimer at the 3ꢁ-end of
the oligonucleotides, exhibited ten folds increase in resistance to exonucleases
(15).
In summary, oligonucleotides containing hydroxamate dimers have been syn-
thesized for the first time and studied for their ability to form stable duplexes. In-
terestingly, oligonucleotides containing hydroxamate dimer 15 not only displayed
a similar or even slightly higher affinity for RNA target than the natural analogues,
but also showed substantial resistance towards 3ꢁ-exonucleases.

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